Summary The goal of this proposal is to determine the molecular basis for epigenome homeostasis by the coordinated activities of histone chaperone and acetyltransferase complexes. The epigenome is maintained in a state of dynamic equilibrium, or epigenome homeostasis, that is required for appropriately regulated gene expression. A breakdown in epigenome homeostasis is a characteristic of aging cells and age-associated diseases such as cancer, and is accompanied by aberrant epigenetic regulation that is often accompanied by loss of histones, aberrant deposition of acetylated histones and cryptic transcription initiation. This proposal will focus on the coordinated activity of two epigenetic regulatory systems, histone chaperones and histone acetyltransferases, which play major roles in maintaining epigenome homeostasis in healthy cells, and show alterations in aging and senescent cells. Specifically, we will study the HIRA/UBN1/CABIN1 (HUC) histone chaperone complex that specifically deposits the H3.3 histone variant into chromatin in a DNA replication-independent and transcription-coupled manner to promote normal transcriptional programs at specific target genes; and the histone acetyltransferases from yeast and human (Sas2/4/5 and hMOF) that places the H4K16 acetylation (H4K16ac) mark, the elevation of which is correlated with aged cells. HUC-mediated H3.3 deposition and H4K16ac is intimately connected in senescent cells, but not proliferating cells. We will also study the Asf1 histone chaperone (from budding yeast and human), which cooperates with the Rtt109 histone acetyltransferase in budding yeast to deposit acetylated H3K56 histones to promote cryptic transcription initiation, and with the HUC complex in human to maintain epigenome homeostasis. Finally, we will develop H4K16ac and HUC-mediated H3.3 deposition inhibitors to counteract cancer-promoting senescence- associated secretory phenotype (SASP). The specific aims are to (1) Determine the molecular basis for H3.3- specific deposition and targeting by HUC to control gene expression in senescent cells, and develop HUC inhibitors to target SASP, (2) Determine the molecular basis for H4K16 acetylation by the human MOF (hMOF) and orthologous budding yeast Sas2/4/5 complex, and develop small molecule H4K16 acetylation pathway inhibitors to promote cellular healthspan, and (3) Determine the molecular basis for how Asf1 cooperates Rtt109 to mediate cryptic transcription initiation in aging cells. These aims will be tightly connected to Berger (Project 2), Adams (Project 1) and Zhang (Project 4) and use Cores B and C. Together, we anticipate that these studies will lead to a molecular understanding of the concerted roles of H4K16 acetylation and H3.3 histone deposition in control of senescence and aging and cryptic transcription initiation; and to the development of small molecule compounds to inhibit H4K16 acetylation and HUC function in aging cells to inhibit SASP and promote healthspan.